Solid state NMR of salivary calculi: Proline-rich salivary proteins, citrate, polysaccharides, lipids, and organic–mineral interactions

Solid state NMR (ssNMR) can characterize mineral (31P) and organic (13C) components of human salivary stones (n = 8). All show apatitic 31P spectra. 13C ssNMR indicates more protein, of more consistent composition, than apatitic uroliths, with signals from Tyr, Phe and His prominent. Citrate and lipid, identified by dipolar dephasing (DD), and polysaccharides are also observable in varying amounts. 13C{31P} rotational echo double resonance (13C{31P} REDOR) identifies carbon atoms in close (< ca. 0.5 nm) proximity to phosphorus and therefore probably binding with mineral. Citrate, sugar and carboxylate signals undergo strong 13C{31P} REDOR, also seen to signals between 50 and 60 ppm, from protein α- carbons and, possibly, phosphoserines and phospholipids, and sometimes to a 35 – 40 ppm envelope containing Asp-Cβ and Glu-Cγ signals. Amino acid analysis indicates high proline and aromatic content. 13C ssNMR and amino acid composition is consistent with preponderance of proline-rich salivary proteins such as statherin.The U.K. EPSRC (Y. L.) and MRC (D. G. R.) for fundingThis is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.crci.2015.07.00

Solid state NMR of isotope labelled murine fur: a powerful tool to study atomic level keratin structure and treatment effects

We have prepared mouse fur extensively $^{13}$C,$^{15}$N-labelled in all amino acid types enabling application of 2D solid state NMR techniques which establish covalent and spatial proximities within, and in favorable cases between, residues. $^{13}$C double quantum-single quantum correlation and proton driven spin diffusion techniques are particularly useful for resolving certain amino acid types. Unlike 1D experiments on isotopically normal material, the 2D methods allow the chemical shifts of entire spin systems of numerous residue types to be determined, particularly those with one or more distinctively shifted atoms such as Gly, Ser, Thr, Tyr, Phe, Val, Leu, Ile and Pro. Also the partial resolution of the amide signals into two signal envelopes comprising of $\alpha$-helical, and $\beta$-sheet/random coil components, enables resolution of otherwise overlapped $\alpha$-carbon signals into two distinct cross peak families corresponding to these respective secondary structural regions. The increase in resolution conferred by extensive labelling offers new opportunities to study the chemical fate and structural environments of specific atom and amino acid types under the influence of commercial processes, and therapeutic or cosmetic treatments.Medical Research Council (Grant ID: RG75828), Engineering and Physical Sciences Research Council (Ph.D. studentships), National Institute of Health Researc

In situ characterization of advanced glycation end products (AGEs) in collagen and model extracellular matrix by solid state NMR

Non-enzymatic glycation of extracellular matrix with (U-13C5)-Dribose- 5-phosphate (R5P), enables in situ 2D ssNMR identification of many deleterious protein modifications and crosslinks, including previously unreported oxalamido and hemiaminal (CH3- CH(OH)NHR) substructures. Changes in charged residue proportions and distribution may be as important as crosslinking in provoking and understanding harmful tissue changes

Introduction to magnetic resonance methods in photosynthesis

Electron paramagnetic resonance (EPR) and, more recently, solid-state nuclear magnetic resonance (NMR) have been employed to study photosynthetic processes, primarily related to the light-induced charge separation. Information obtained on the electronic structure, the relative orientation of the cofactors, and the changes in structure during these reactions should help to understand the efficiency of light-induced charge separation. A short introduction to the observables derived from magnetic resonance experiments is given. The relation of these observables to the electronic structure is sketched using the nitroxide group of spin labels as a simple example

Ucma/GRP inhibits phosphate-induced vascular smooth muscle cell calcification via SMAD-dependent BMP signalling

Vascular calcification (VC) is the process of deposition of calcium phosphate crystals in the blood vessel wall, with a central role for vascular smooth muscle cells (VSMCs). VC is highly prevalent in chronic kidney disease (CKD) patients and thought, in part, to be induced by phosphate imbalance. The molecular mechanisms that regulate VC are not fully known. Here we propose a novel role for the mineralisation regulator Ucma/GRP (Upper zone of growth plate and Cartilage Matrix Associated protein/Gla Rich Protein) in phosphate-induced VSMC calcification. We show that Ucma/GRP is present in calcified atherosclerotic plaques and highly expressed in calcifying VSMCs in vitro. VSMCs from Ucma/GRP(-/-) mice showed increased mineralisation and expression of osteo/chondrogenic markers (BMP-2, Runx2, beta-catenin, p-SMAD1/5/8, ALP, OCN), and decreased expression of mineralisation inhibitor MGP, suggesting that Ucma/GRP is an inhibitor of mineralisation. Using BMP signalling inhibitor noggin and SMAD1/5/8 signalling inhibitor dorsomorphin we showed that Ucma/GRP is involved in inhibiting the BMP-2-SMAD1/5/8 osteo/chondrogenic signalling pathway in VSMCs treated with elevated phosphate concentrations. Additionally, we showed for the first time evidence of a direct interaction between Ucma/GRP and BMP-2. These results demonstrate an important role of Ucma/GRP in regulating osteo/chondrogenic differentiation and phosphate-induced mineralisation of VSMCs.NWO ZonMw [MKMD 40-42600-98-13007]; FCT [SFRH/BPD/70277/2010]info:eu-repo/semantics/publishedVersio

Phospholipid headgroup dynamics in DOPG-d5-cytochrome c complexes as revealed by 2H and 31P NMR: the effects of a peripheral protein on collective lipid fluctuations.

The dynamics of the glycerol headgroup of dioleoylphosphatidylglycerol (DOPG) in hydrated bilayers were studied by 2H and 31P NMR spectroscopy, and the effects of binding a peripheral protein, cytochrome c, were evaluated. The fast headgroup segmental motions (tau c, 10(-10)-(-13) s) of DOPG in fully hydrated bilayers were not affected upon binding of cytochrome c, as evaluated by the spin-lattice (T1) relaxation of deuterons in the DOPG glycerol headgroup. In contrast, the spin-spin (T2e) relaxation is strongly affected, indicating that slow cooperative bilayer motions (tau c, 10(-3)-10(-6) s) are enhanced upon the interaction with cytochrome c, 2H and 31P NMR spectral lineshape analysis reveal details of the nature of these motions. The importance of these effects are discussed in terms of a possible mechanism for modulating membrane-associated processes

Dehydration and crystallization of amorphous calcium carbonate in solution and in air

The mechanisms by which amorphous intermediates transform into crystalline materials are poorly understood. Currently, attracting enormous interest is the crystallization of amorphous calcium carbonate, a key intermediary in synthetic, biological and environmental systems. Here we attempt to unify many contrasting and apparently contradictory studies by investigating this process in detail. We show that amorphous calcium carbonate can dehydrate before crystallizing, both in solution and in air, while thermal analyses and solid-state nuclear magnetic resonance measurements reveal that its water is present in distinct environments. Loss of the final water fraction-comprising less than 15% of the total-then triggers crystallization. The high activation energy of this step suggests that it occurs by partial dissolution/recrystallization, mediated by surface water, and the majority of the particle then crystallizes by a solid-state transformation. Such mechanisms are likely to be widespread in solid-state reactions and their characterization will facilitate greater control over these processes

Collagen labelling with an azide-proline chemical reporter in live cells

We have developed a strategy for selective imaging of collagen in live foetal ovine osteoblasts. Our approach involves the incorporation of an azide-tagged proline in the biosynthesis of collagen followed by labelling using a strain-promoted [3+2] azide-alkyne cycloaddition reaction. This journal i